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Both PIGA and PIGL mutations cause GPI-a deficient isolates in the Tk6 cell line
Janice A Nicklas1, Elizabeth W Carter2, Richard J Albertini3
1Department of Pediatrics, University of Vermont College of Medicine, Burlington, Vermont.
Environmental and Molecular Mutagenesis
|May 14, 2015
Summary
Molecular analysis revealed that glycosylphosphatidylinositol anchor (GPI-a) deficiency in TK6 cells is often caused by PIGL gene deletions, not just PIGA mutations. This finding highlights a dual-gene mutation assay for GPI-a deficiency.
Area of Science:
- Genetics
- Molecular Biology
- Cell Biology
Background:
- Glycosylphosphatidylinositol anchor (GPI-a) deficiency is a condition affecting cell surface protein anchoring.
- TK6 cell line is a common model for studying genetic mutations and cellular deficiencies.
- X-linked PIGA mutations are a known cause of GPI-a deficiency, but other genetic factors can be involved.
Purpose of the Study:
- To investigate the molecular basis of GPI-a deficiency in TK6 cell line isolates, particularly those without PIGA mutations.
- To identify the genes and mutation types responsible for the remaining GPI-a deficient isolates.
- To evaluate the TK6 cell line as a model for detecting multiple gene mutations simultaneously.
Main Methods:
- Molecular analysis of proaerolysin-selected GPI-a deficient TK6 cell isolates.
- Ethyl methane sulfate (EMS) treatment to induce mutations.
- Real-time analysis of 25 autosomal GPI-a pathway genes.
- Deletion analysis (including breakpoint sequencing) and point mutation detection in the PIGL gene.
- Analysis of the TK6 parent cell line for pre-existing deletions.
Main Results:
- X-linked PIGA mutations were infrequent in spontaneous isolates but increased after EMS treatment.
- A significant number of GPI-a deficient isolates lacked PIGA mutations and showed absent PIGL mRNA.
- Homozygous deletions in the 5' region of PIGL (17p12-p22), extending through NCOR1 and TTC19, were identified.
- The TK6 parent cell line possessed a hemizygous deletion in the same chromosomal region.
- Point mutations in the PIGL gene were also observed in isolates lacking PIGA mutations or homozygous PIGL deletions.
Conclusions:
- GPI-a deficiency in TK6 cells can result from mutations in autosomal genes, notably PIGL, in addition to X-linked PIGA.
- The TK6 cell line is susceptible to large deletions in the 17p12-p22 region affecting PIGL.
- The TK6 cell line serves as a valuable model for simultaneously detecting both point and deletion mutations in multiple genes involved in GPI-a biosynthesis.
